Enhancing the biophysical properties of mRFP1 through incorporation of fluoroproline

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• 作者：Kanagavel Deepankumar ; Saravanan Prabhu Nadarajan ; Niraikulam Ayyadurai ; Hyungdon Yun
• 关键词：NCAA ; non-canonical amino acid incorporation ; FP ; fluoroproline ; (4R)-FP ; (2S ; 4R)-4-fluoroproline ; (4S)-FP ; (2S ; 4S)-4-fluoroproline ; MM ; minimal media ; GdmCl ; guanidine hydrochloride
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：1 November, 2013
• 年：2013
• 卷：440
• 期：4
• 页码：509-514
• 全文大小：1127 K

文摘

Here we enhanced the stability and biophysical properties of mRFP1 through a combination of canonical and non-canonical amino acid mutagenesis. The global replacement of proline residue with (2S, 4R)-4-fluoroproline [(4R)-FP] into mRFP1 led to soluble protein but lost its fluorescence, whereas (2S, 4S)-4-fluoroproline [(4S)-FP] incorporation resulted in insoluble protein. The bioinformatics analysis revealed that (4R)-FP incorporation at Pro63 caused fluorescence loss due to the steric hindrance of fluorine atom of (4R)-FP with the chromophore. Therefore, Pro63 residue was mutated with the smallest amino acid Ala to maintain non coplanar conformation of the chromophore and helps to retain its fluorescence with (4R)-FP incorporation. The incorporation of (4R)-FP into mRFP1-P63A showed about 2-3-fold enhancement in thermal and chemical stability. The rate of maturation is also greatly accelerated over the presence of (4R)-FP into mRFP1-P63A. Our study showed that a successful enhancement in the biophysical property of mRFP1-P63A[(4R)-FP] using non-canonical amino acid mutagenesis after mutating non-permissive site Pro63 into Ala.